INTRODUCTION: Translational control of cytokine production in endotoxin (LPS)-stimulated macrophages is poorly characterized but likely important. An early step in protein translation is engagement of mRNA by eukaryotic initiation factor 4E (eIF-4E). Translation initiation can be prevented by small 4E-binding proteins (4E-BP1 or PHAS-I) which must be phosphorylated in order to disengage eIF-4E. We examined whether LPS alters 4E-BP1 phosphorylation in macrophages. MATERIALS AND METHODS: Elicited rat peritoneal macrophages and Raw 264.7 macrophages were treated with signal transduction inhibitors and then LPS. Cells were harvested and equal protein amounts were electrophoresed (SDS-PAGE). Western blots (WB) were developed with 4E-BP1 antibody. Alternatively cell lysates were exposed to 7-methyl GTP Sepharose beads in order to isolate the cap-binding protein eIF-4E. The relative amounts of 4E-BP1 associated with eIF-4E were then determined by WB. RESULTS: Macrophage 4E-BP1 is phosphorylated upon stimulation by LPS as evidenced by the appearance of a more slowly migrating gamma (hyperphosphorylated) band on gel electrophoresis. Inhibition of both the p42/p44 MAPK pathway (PD 98059) and the p38 MAPK pathway (SB 203580) failed to alter LPS-induced 4E-BP1 phosphorylation. Rapamycin (FRAP/mTOR inhibitor) blocked 4E-BP1 phosphorylation causing a predominance of the alpha (hypophosphorylated) band. This was confirmed further by 7-methyl-GTP Sepharose isolation of eIF-4E with which 4E-BP1 coprecipitates. CONCLUSION: LPS stimulates 4E-BP1 phosphorylation in macrophages through FRAP/mTOR signaling. This pathway may contribute to the translational control of cytokine gene expression in macrophages. Copyright 2001 Academic Press.
INTRODUCTION: Translational control of cytokine production in endotoxin (LPS)-stimulated macrophages is poorly characterized but likely important. An early step in protein translation is engagement of mRNA by eukaryotic initiation factor 4E (eIF-4E). Translation initiation can be prevented by small 4E-binding proteins (4E-BP1 or PHAS-I) which must be phosphorylated in order to disengage eIF-4E. We examined whether LPS alters 4E-BP1 phosphorylation in macrophages. MATERIALS AND METHODS: Elicited rat peritoneal macrophages and Raw 264.7 macrophages were treated with signal transduction inhibitors and then LPS. Cells were harvested and equal protein amounts were electrophoresed (SDS-PAGE). Western blots (WB) were developed with 4E-BP1 antibody. Alternatively cell lysates were exposed to 7-methyl GTPSepharose beads in order to isolate the cap-binding protein eIF-4E. The relative amounts of 4E-BP1 associated with eIF-4E were then determined by WB. RESULTS: Macrophage 4E-BP1 is phosphorylated upon stimulation by LPS as evidenced by the appearance of a more slowly migrating gamma (hyperphosphorylated) band on gel electrophoresis. Inhibition of both the p42/p44 MAPK pathway (PD 98059) and the p38 MAPK pathway (SB 203580) failed to alter LPS-induced 4E-BP1 phosphorylation. Rapamycin (FRAP/mTOR inhibitor) blocked 4E-BP1 phosphorylation causing a predominance of the alpha (hypophosphorylated) band. This was confirmed further by 7-methyl-GTPSepharose isolation of eIF-4E with which 4E-BP1 coprecipitates. CONCLUSION:LPS stimulates 4E-BP1 phosphorylation in macrophages through FRAP/mTOR signaling. This pathway may contribute to the translational control of cytokine gene expression in macrophages. Copyright 2001 Academic Press.
Authors: Valérie Schaeffer; Saman Arbabi; Iris A Garcia; Megan L Knoll; Joseph Cuschieri; Eileen M Bulger; Ronald V Maier Journal: J Surg Res Date: 2010-06-09 Impact factor: 2.192